DESCRIPTION (Applicant's Description): The Wnt-beta-catenin signaling pathway is a key regulatory component of early embryonic development, cell fate decisions, and cell growth regulation. Improper regulation of beta-catenin levels and nuclear localization can result in severe embryonic defects, failure to form particular tissues, and a loss of cellular growth control leading to cancer. This research proposal focuses on the beta-catenin-mediated signaling required for formation of the dorsal embryonic signaling center known as the "organizer." A zebrafish recessive maternal effect mutant isolated in our laboratory, ichabod, is severely impaired in organizer formation and function. ichabod mutant embryos fail to form anterior and dorsal tissues and are the most severely ventralized embryos of known zebrafish mutants. We have demonstrated that an early defect in ichabod embryos is the failure of beta-catenin to be localized in nuclei on the dorsal side of the embryo. Our recent results indicate that the lesion in the mutant affects nuclear localization in a manner independent of both GSK3 function and recruitment of beta-catenin for degradation. ichabod may thus function in a part of the signaling pathway that is not well understood at present. We propose to: (1) identify the gene product encoded by the ichabod gene, (2) isolate additional alleles of the ichabod gene and identify genes that interact genetically with ichabod, (3) further characterize the impairment of beta-catenin nuclear localization in mutant embryos and determine whether stability of beta-catenin is in fact unperturbed, and (4) characterize the effect of the ichabod mutation on early steps in dorsoventral and anteroposterior axis formation. The optical clarity of the zebrafish embryo provides a particularly suitable embryonic system to use GFP assays to study nuclear localization of beta-catenin and to determine the sites of beta-catenin induced gene activation. These studies should increase our understanding of the control of nuclear localization of beta-catenin and also allow an analysis of the initial patterning defects that result from failure to form the early dorsal signaling center.